EP3698628A1 - Stabilisierung von vollblutproben - Google Patents

Stabilisierung von vollblutproben Download PDF

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EP3698628A1
EP3698628A1 EP20150503.9A EP20150503A EP3698628A1 EP 3698628 A1 EP3698628 A1 EP 3698628A1 EP 20150503 A EP20150503 A EP 20150503A EP 3698628 A1 EP3698628 A1 EP 3698628A1
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Prior art keywords
blood
sample
platelet
cells
whole blood
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French (fr)
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Rebecca SANDLIN
Keith Wong
Shannon TESSIER
Mehmet Toner
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General Hospital Corp
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General Hospital Corp
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes

Definitions

  • This invention relates to methods for stabilizing blood samples, e.g., clinical blood samples, for storage.
  • Peripheral blood is the most frequently accessed tissue in the clinic, and the isolation of blood-borne cells is of broad clinical and scientific importance in hematology, transfusion, immunology, regenerative medicine, and oncology. Recent developments in microengineering have greatly advanced our capabilities in isolating pure populations of cells and performing high-throughput, multidimensional assays (1).
  • the rapidly growing field of blood-on-a-chip technologies has expanded into applications ranging from T cell isolation for HIV disease monitoring (2) and multiplexed detection of cytokine secretion (3); gene expression profiling of neutrophils in trauma and burn patients (4); enrichment of CD34+ hematopoietic stem cells (5); to minimally invasive detection of nucleated red blood cells (RBCs) from maternal blood (6) as well as rare circulating tumor cells for cancer diagnosis (7) and identification of druggable mutations (8).
  • Red blood cell rouleaux formation may trap rare cells.
  • microfluidic sorting technologies which are essential for efficient cell sorting are compromised by echinocytes (aged red blood cells that form spiculations), platelet activation and clotting, as well as cellular aggregation.
  • hypothermic temperature ranges which can otherwise effectively suppress biochemical reactions and degradation processes have been considered incompatible, due mainly to cold-induced platelet activation.
  • whole blood is typically stored in room temperature and processed into various components for specialized storage within 24 hours (9).
  • clinically relevant assays such as sequencing and expression profiling are best performed in large medical centers or diagnostic laboratories, the logistical needs of blood storage and transportation impose severe limitations to the dissemination of next-generation blood-based medical technologies.
  • CTCs circulating tumor cells
  • Advanced microfluidic technologies have enabled isolation of these extremely rare cells (one in a billion blood cells) from peripheral blood samples of cancer patients, and significant progress has been made in using these cells and their molecular signature for diagnosis, prognosis, identification of druggable mutations, as well as generation of patient-specific models for drug screening.
  • these downstream assays are critically dependent upon the isolation of viable, unfixed CTCs that retain the molecular information and cellular function. Degradation of blood not only restricts molecular analysis of CTCs, but also interferes with the precise microfluidic isolation of these extremely rare cells.
  • Described herein are several methods, each of which can be used singly or in combination, to preserve whole blood samples for numerous clinical applications. Exemplary applications include but are not limited to: enrichment of leukocyte subtypes such as T-cells or neutrophils for cytokine and immuno-assays; isolation of progenitor cells from cord blood or peripheral blood for transplantation; isolation of fetal cells from the maternal blood for diagnosis; and sorting of circulating tumor cells for cancer detection and therapy.
  • leukocyte subtypes such as T-cells or neutrophils for cytokine and immuno-assays
  • isolation of progenitor cells from cord blood or peripheral blood for transplantation
  • isolation of fetal cells from the maternal blood for diagnosis
  • sorting of circulating tumor cells for cancer detection and therapy.
  • the invention provides methods for stabilizing a sample of whole blood.
  • the methods include obtaining a sample of whole blood from a subject, and introducing to the sample Ficoll 70 to produce 2-20% (w/v) Ficoll 70 in the sample.
  • the methods include obtaining a sample of whole blood from a subject, and introducing to the sample a caspase inhibitor, and optionally a preservative formulation.
  • the methods include obtaining a sample of whole blood from a subject, and introducing to the sample a preservative formulation, wherein the preservative formulation comprises 48 mM HEPES, 0.44 mM adenine, 6.75 mM mannitol, 0.77 mM N-acetyl-L-cysteine, and 8.5 mM NaCl.
  • the methods include obtaining a sample of whole blood from a subject; and introducing a platelet inhibitor (PI) to the sample.
  • PI platelet inhibitor
  • Also provided are methods for stabilizing a sample of whole blood comprising obtaining a sample of whole blood from a subject, and introducing to the sample one or more of: Ficoll 70 to produce 2-20% (w/v) Ficoll 70 in the sample; a caspase inhibitor; a preservative formulation that comprises 48 mM HEPES, 0.44 mM adenine, 6.75 mM mannitol, 0.77 mM N-acetyl-L-cysteine, and 8.5 mM NaCl; and/or a platelet inhibitor (PI).
  • PI platelet inhibitor
  • Ficoll 70 is added to produce at least 10% Ficoll 70 in the sample.
  • the caspase inhibitor is Q-VD-OPh ((3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid), Z-VAD-FMK (methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoate), Q-VD(OMe)-OPh ((S)-methyl 5-(2,6-difluorophenoxy)-3-((S)-3-methyl-2-(quinoline-2-carboxamido)butanamido)-4-oxopentanoate), or Boc-D-fmk (methyl 5-fluoro-3-[(2-methylpropan-2-
  • the preservative formulation comprises 24 - 48 mM HEPES, 0.11 - 0.44 mM adenine, 2.25 - 6.75 mM mannitol, 0.39 - 1.54 mM N-acetyl-L-cysteine, 0 - 13.5 mM dextrose, and 0 - 17 mM NaCl.
  • the preservative formulation comprises 48 mM HEPES, 0.44 mM adenine, 6.75 mM mannitol, 0.77 mM N-acetyl-L-cysteine, and 8.5 mM NaCl.
  • the blood is stabilized for storage at 20-25°C.
  • the blood is maintained or stored for 72-96 hours, e.g., at 20-25°C.
  • the PI is ticagrelor, cilostazol, prasugrel, dipyridamole, prasugrel, Tirofiban, eptifibatide, clopidogrel, or KF38789.
  • the PI is added to the sample to achieve a final concentration of from 0.01-100 ug/mL, e.g., from 0.01-1 ug/ml, e.g., from 0.01-0.5 ug/mL.
  • the blood is stabilized for storage at 2-25°C. In some embodiments, is stabilized for storage at 4°C. In some embodiments, the methods include maintaining the sample at 4°C. The method of claims 4, 5, or 13-16, wherein the sample is maintained or stored at 2-25°C for at least 24, 36, 48, 72, or 96 hours. For example, the samples can be held for 72 hours (for neutrophil stabilization) and at least 96 hours (for erythrocyte stabilization)
  • introducing to the sample can mean adding something to the sample, or adding the sample to something (e.g., putting the sample into a tube that already includes the additive).
  • the present methods are particularly useful for samples for clinical and laboratory diagnostics, such as microfluidic evaluation of whole blood, and neutrophil migration assays that can indicate the functioning of the immune system (e.g., in diagnosis of sepsis).
  • the optimized preservative with optional caspase inhibitor can be selected.
  • the optimized preservative with optional caspase inhibitor can be selected, with platelet inhibitors also included.
  • leukocyte subtypes such as T-cells or neutrophils for cytokine and immuno-assays
  • Blood sedimentation can be understood in terms of simple physics.
  • ⁇ p is the density of sphere
  • ⁇ f is the density of fluid
  • ⁇ is the fluid viscosity
  • g gravity
  • R is the radius of the sphere. Therefore, settling speed is proportional to the density difference (between the sphere and the fluid) and the square of the radius, and is inversely proportional to fluid viscosity. This equation explains why red blood cell aggregation (increased R) increases the erythrocyte settling rate (ESR).
  • ESR erythrocyte settling rate
  • Ficoll polysaccharide Ficoll
  • Polymers with similar physiochemical properties such as dextran and polyethylene glycol, e.g., dextran 40kDa polymers, can also be used.
  • dextran 40kDa polymers can also be used.
  • Ficoll polymers into blood is simple and is compatible with common assays for leukocyte enrichment. Physical stabilization inhibited red blood cell aggregation, echinocyte formation, maintained leukocyte viability, and prevented NETosis of neutrophils.
  • Red blood cells which constitute 99% of the total volume of all blood cells, retained their biconcave morphology in the presence of F70. This result should benefit applications such as size-based cell sorting (e.g., filtration) or microfluidic processes that rely on hydrodynamic properties of cells such as deterministic lateral displacement (30).
  • Leukocytes in particular the fragile neutrophils displayed superior integrity and decreased apoptosis.
  • the inhibition of NET formation has important implications in blood storage. It is known that activated neutrophils release chromatin fibers mixed with neutrophil enzymes to form NETs (31). NETs protects against infection but may also promote thrombosis (32) and autoimmune reactions (33).
  • NETs components such as DNA, neutrophil elastase, and myeloperoxidase-induces collateral damage to other cells in whole blood and accelerate the degradation of the entire blood sample. Therefore, the preservation of all blood cells is critical even in applications where the cells of interest are non-hematologic. For instance, the sorting and analysis of rare circulating tumor cells (1 in 10 9 blood cells) holds great potential as a non-invasive liquid biopsy for the clinical management of cancer (35). Degradation of hematologic cells not only cause collateral damage to the rare circulating tumor cells, but also negatively impact cell sorting technologies that rely on defined biological and physical properties of blood cells.
  • Ficoll polymers may provide an opportunity to improve existing procedures and assays. For instance, immunological assays are sometimes performed with a 24-hour delay to account for specimen shipping. Short-term storage of cord blood for up to 48 hours prior to cryopreservation is also a common practice. Within these time frames, the degrading granulocytes (in which >90% are neutrophils) have been found to negatively impact T-cell assays (36, 37). The ability of Ficoll to maintain neutrophil integrity especially within the shorter time frames ( Fig. 5 ) may find immediate use in similar applications.
  • an optimized preservative formulation was developed that optionally includes a caspase inhibitor.
  • This formulation is particularly suited for clinical samples, and for all samples that are to be maintained at ambient (e.g., 20-25°C) temperatures.
  • pan-caspase inhibitors include Q-VD-OPh ((3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid), Z-VAD-FMK (methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl] amino]propanoyl]amino]-4-oxopentanoate), Q-VD(OMe)-OPh ((S)-methyl 5-(2,6-difluorophenoxy)-3-((S)-3-methyl-2-(quinoline-2-carboxamido)butanamido)-4-oxopentanoate), or Boc-D-fmk (methyl 5-fluoro-3
  • the preservative formulation can be, e.g., CS-18 shown below, and can include Q-VD-OPh, e.g., at 2-10 uM, e.g., about 5 uM, 24 - 48 mM HEPES, 0.11 - 0.44 mM adenine, 2.25 - 6.75 mM mannitol, 0.39 - 1.54 mM N-acetyl-L-cysteine, 0-13.5 mM dextrose, and 0 - 17 mM NaCl.
  • a preferred formulation is 48 mM HEPES, 0.44 mM adenine, 6.75 mM mannitol, 0.77 mM N-acetyl-L-cysteine, and 8.5 mM NaCl.
  • a platelet inhibitor is added to the sample before the samples are cooled and kept cold, e.g., at 2-25°C.
  • the platelet inhibitor prevents platelets from activating and aggregating, which interferes with isolation technologies including, but not limited to, antibody-based cell enrichment and microfluidic blood cells sorting,
  • a number of platelet inhibitors are known in the art, including cyclooxygenase inhibitors (e.g., acetylsalicylic acid and triflusal (Disgren)); adenosine diphosphate (ADP) receptor inhibitors (e.g., clopidogrel (Plavix), Prasugrel (Effient), ticagrelor (Brilinta), or ticlopidine (Ticlid)); Phosphodiesterase inhibitors (e.g., cilostazol (Pletal)); Protease-activated receptor-1 (PAR-1) antagonists (e.g., vorapaxar (Zontivity)
  • the platelet inhibitor is a Glycoprotein IIB/IIIA inhibitor, e.g., Tirofiban, roxifiban, orbofiban, eptifibatide, or abciximab.
  • a sufficient amount of the platelet inhibitor is added to the sample to produce a dosage ranging from 0.01-100 ug/ml, e.g., from 0.01-1 ug/ml, e.g., from 0.01-0.5 ug/mL.
  • Blood samples were obtained from healthy volunteers or purchased from Research Blood Components (Brighton, MA). All blood samples were drawn into Acid Citrate Dextrose-A (ACD-A) tubes (BD Vacutainer; 8.5 mL) and used within four hours.
  • ACD-A Acid Citrate Dextrose-A
  • the erythrocyte settling rate (ESR) assay was performed with 1.3 mL of whole blood or Ficoll-blood using pipets that conform to the dimensions of the standardized Westergren method (Dispette 2; Fisherbrand). The ESR in millimeters was recorded at 24, 48, and 72 hours.
  • Couette rheometry was performed using a TA Instruments Discovery HR-3 rheometer with a steel double-wall concentric cylinder geometry. 8.5 mL of sample was used for each experiment. Data points were acquired at shear rates ranging from 0.1/s to 1000/s, at 5 points per decade.
  • echinocytes For the enumeration of echinocytes, samples were gently mixed before a drop ( ⁇ 10 ⁇ L) was transferred to a glass slide, smeared, and imaged using phase-contrast microscopy at 40 ⁇ using an EVOS FL Cell Imaging System (Life Technologies). About 100 random RBCs were counted per sample and echinocytes were identified by their distinct spiculations.
  • Red blood cell lysis was performed using Red Blood Cell Lysis Solution (Miltenyi Biotec). After lysis, cells were spun down at 300 ⁇ g, washed with 10 mL RoboSep buffer (Miltenyi Biotec), spun again, resuspend in 600 ⁇ L RoboSep buffer, and counted using a Beckman Z2 Coulter Counter.
  • Neutrophil enrichment was performed using the EasySep Human Neutrophil Enrichment Kit (Stemcell Technologies) according to manufacturer's protocol. Briefly, the depletion antibody cocktail was mixed with the enriched leukocytes (obtained by RBC lysis) followed by incubation with magnetic particles.
  • the EasySep Magnet was then used to immobilize unwanted cells as the label-free neutrophils were poured into another conical tube. Enriched neutrophils were then re-spun and resuspended in 1 mL RPMI media containing 0.3% BSA and 10 mM HEPES, counted, and stained for imaging flow cytometry.
  • Imaging flow cytometry was performed using the ImageStream X Mark II imaging flow cytometer (Amnis Corporation) equipped with a 40 ⁇ objective, 6 imaging channels, and 405 nm, 488 nm, and 642 lasers.
  • the enriched leukocytes were resuspended in 0.1% BSA in HEPES buffered saline after RBC lysis and stained with the following antibodies and stains where applicable: DRAQ5 (1 ⁇ M; Cell Signaling Technologies), Sytox Blue (1 ⁇ M; Life Technologies), CellEvent Caspase-3/7 Green Detection Reagent (0.75 ⁇ M; Life Technologies), FITC-conjugated CD45 antibody (1:500; clone 5B1; Miltenyi Biotec), PE-conjugated CD66b antibody (1:125; clone G10F5; Stemcell Technologies), and PE-Cy7-conjugated CD16 antibody (1:200 or 1:333; clone 3G8
  • Single cells were gated using the nuclear marker DRAQ5.
  • Neutrophils were identified by the dual positivity of CD66b and CD16.
  • DRAQ5 (1 ⁇ M; Cell Signaling Technologies)
  • VioBlue-conjugated CD45 antibody (1:100; clone 5B1; Miltenyi Biotec)
  • Alexa Fluor 488-conjugated CD11b antibody (1:500; clone ICRF44; Stemcell Technologies
  • PE-conjugated CD66b antibody (1:125; clone G10F5; Stemcell Technologies
  • PE-Cy7-conjugated CD16 antibody (1:333; clone 3G8; BD Biosciences).
  • the slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG (both 1:500; Life Technologies) for 45 minutes at room temperature, rinsed with PBS, and mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Imaged were captured with a QImaging Retiga 2000R camera using a Nikon S Plan Fluor ELWD 60x/0.70 objective on a Nikon Eclipse 90i microscope.
  • Ficoll polymers stabilized whole blood we characterized their rheological properties which are relevant in blood settling, namely, viscosity, density, and cell aggregation.
  • Whole blood exhibited shear-thinning behavior, with viscosity decreasing from 51.9 ⁇ 18.9 cP at a shear rate of 0.1/s to 4.1 ⁇ 0.4 cP at a shear rate of 1000/s ( Fig. 2A ).
  • the densities of WB, 5% F70, 10% F70, and 15% F70 were 1.047 ⁇ 0.009 g/mL, 1.051 ⁇ 0.007 g/mL, 1.063 ⁇ 0.006 g/mL, and 1.071 ⁇ 0.006 g/mL respectively ( Fig. 2B ; p ⁇ 0.0001, 1-way ANOVA with posttest for linear trend).
  • echinocytes A distinctive feature of RBC aging is characterized by the loss of biconcave disc morphology and emergence of spiculations, termed echinocytes ( Fig. 3A ).
  • Fig. 3A The percentage of echinocytes as a result of storage in settled whole blood or Ficoll-stabilized blood ( Fig, 3B ).
  • Echinocyte levels in whole blood increased from 6.6 ⁇ 11.6 % at 0 hour to 52.2 ⁇ 25.4 %, 63.2 ⁇ 25.9 %, and 78.8 ⁇ 21.1 % at 24, 48, and 72 hours respectively.
  • NETs neutrophil extracellular traps
  • Example 2 Erythrocyte and Leukocyte stabilization in whole blood under ambient conditions
  • CS-original** (cocktail solution original)
  • CS was prepared in water and added to blood collected in ACD anticoagulant tubes at a ratio of 17:3 blood:preservative.
  • concentration noted in Table 1 was the final concentration of preservative component in the blood.
  • samples are gassed with blood gas (5% O2, 5% CO2) and sealed. Samples were then stored at 21°C for the desired amount of time (24-96 h). After the storage period, a 20 mM solution of adenosine is added to blood so that the final concentration is 2 mM.
  • Neutrophils were isolated by negative selection from healthy donor whole blood using the recommended protocol and reagents from the Human Neutrophil Enrichment Kit (StemCell Technologies; Vancouver, British Columbia, Canada). Yield was determined by counting particles from 8-30 ⁇ m in diameter using a Coulter Counter Z1 (Beckman Coulter; Brea, California). Neutrophils were stored in IMDM + 20% FBS and were either untreated (control) or treated with 1.25 ⁇ L/mL DMSO (Sigma-Aldrich; St.
  • Example 2.1 Taguchi optimization of preservative formulation for patient erythrocyte stabilization.
  • the CS-original formulation designed to stabilize erythrocytes in healthy donor whole blood, was not effective when tested against metastatic breast cancer patient samples, which degrade rapidly.
  • a modified Taguchi table (Table 1 above) was therefore constructed to guide the optimization of the CS formulation. The most promising formulation to date was condition 18 from the Taguchi table.
  • the CS-Original formulation was sufficient for stabilizing erythrocytes from healthy donor samples (stored under ambient conditions as whole blood), but it is not ideal for stabilizing patient samples, which may degrade more rapidly.
  • modified Taguchi methods for design of experiments combined with a post-storage revitalization step (4h incubation with adenosine) the formulation was optimized, resulting in superior protection of samples stored for 72 h (Condition 18, shown in Figure 6 ).
  • Example 2.2 Extensive CD45 biomarker degradation is observed in leukocytes stored under ambient conditions.
  • FIG. 7 shows that the leukocytes stored under ambient conditions in CS-Original underwent significant decreases in CD45 expression compared to freshly examined leukocytes.
  • the apoptosis inhibitors Q-VD-OPh and Boc-D-fmk were selected based on their broad-spectrum caspase inhibitory activity.
  • Q-VD-OPh has previously been reported as a powerful apoptosis inhibitor effective at low concentrations (5 uM).
  • Figures 8A-I reveal, Q-VD-OPh was highly effective at apoptosis inhibition within the very sensitive neutrophil population, and it also acted to extend viability from ⁇ 24 h to 96 h under ambient storage conditions (it is worth noting that the control condition itself also has stabilizing effects on neutrophils, as experiments with HBSS or IMDM only resulted in more rapidly degrading neutrophils which were stable for ⁇ 24 h).
  • the Q-VD-OPh effectively preserved CD45 biomarker expression. Specifically as shown in Figure 8C , after 96 h ambient storage the neutrophils are nearly indistinguishable from those examined immediately after collection. Alternatively, neutrophils from the same donor stored under control conditions, had undergone significant degradation at 96 h ( Figure 8B ).
  • fMLP a neutrophil chemoattractant
  • the Q-VD-OPh treated neutrophils were stable under ambient storage conditions for 72-96 h. Furthermore, the viability and CD45 biomarker expression was stabilized for up to 96 h, a significant improvement upon untreated neutrophils.
  • the Example describes the development of new strategies for the stabilization of whole blood. These methods can be used in samples intended for use in isolation of Circulating Tumor Cells (CTC), e.g., using a microfluidic device known as the CTC-iChip.
  • CTC Circulating Tumor Cells
  • the successful analysis of patient samples currently requires freshly collected blood as a result of the rapid deterioration of patient blood ex vivo. Consequently, the stabilization of whole blood would allow patient samples to be preserved during shipment to centralized facilities equipped for effective CTC isolation.
  • Platelet inhibitors used include: Tirofiban (Sigma), Eptifibatide (Tocris), Clopidogrel (Sigma), KF38789 (Tocris).
  • Tirofiban Sigma
  • Eptifibatide Tocris
  • Clopidogrel Sigma
  • KF38789 Tocris
  • Peripheral blood smears were prepared manually using a wedge technique according to standard procedures. Blood was pipetted ( ⁇ 6 ⁇ l) onto a slide, evenly smeared using a second slide (at a 30-45° angle), and air dried. Blood smears were fixed with methanol (100%), stained with Giemsa-Wright stain (Sigma) for 30 seconds, and quickly transferred to distilled water before air drying. Slides were mounted with Permount before imaging using a Nikon Eclipse 90i microscope, a Nikon 100 ⁇ Apo VC 100x/1.40 oil objective, and Nikon DS-Ri1 color camera (12-bit; 1280 ⁇ 1024 resolution). About 100 random RBCs were counted per sample and classified as either echinocytes (based on distinct thorny projections) or healthy RBCs.
  • Platelet activation and cell viability were determined using the Image Stream Mark II imaging flow cytometer (Amnis Corporation) equipped with a 40 ⁇ objective and the 405, 488, 642 nm lasers.
  • 1 ⁇ l of whole blood was combined with PBS and fluorescently labeled antibodies as follows; Pacific Blue conjugated CD41 (1:150; clone HIP8; BioLegend), FITC conjugated PAC-1 (1:10; clone PAC-1 (RUO (GMP)); BD Pharmingen), PE conjugated CD62P (1:100; clone AK-4; BD Pharmingen), APC conjugated CD63 (1:10; clone H5C6; Biolegend), PE/Cy7 conjugated CD45 (1:100; clone HI30; Abcam).
  • RPMI containing Hepes
  • fluorescently labeled antibodies/viability stains as follows; calcein blue AM (10 ⁇ M; Life Technologies), CellEventTM Caspase-3/7 Green (5 ⁇ M; Life Technologies), PE conjugated CD66b (1:125, clone g10f5, Stemcell Technologies), PE-cf594 conjugated CD45 (1:400, clone HI30, BD Pharmingen).
  • LNCaP prostate adenocarcinoma cells
  • lines derived from breast cancer patients were spiked into whole blood at 300,000 cells/mL.
  • the processing of rare cells for imaging flow cytometry followed the same procedure as described above for white blood cell viability with the following exceptions; PE conjugated EpCAM (1:250, clone VU1D9, Cell Signaling Technology).
  • Lyophilized collagen (soluble calf skin), thrombin, and ristocetin were obtained from Chrono-Log Corporation (Havertown, PA) and reconstituted using sterile distilled water. Aggregation experiments were performed in whole blood using electrical impedance on a two channel Chrono-Log 700 Series Whole Blood/Optical Lumi-Aggregometer and analyzed using AGGRO/LINK8 software. Whole blood was incubated at 37°C for 5 min and each sample was run for 10 min after addition of the agonist with a stir bar speed of 1200 rpm. Agonist concentrations for collagen, ristocetin, and thrombin were 2 ug/mL, 1 mg/mL, 1 Unit/mL, respectively. Collected data included maximal aggregation (%), slope of the aggregation curve, area under the curve at 6 min as well as elapsed time between addition of agonist and onset of aggregation (lag phase).
  • Cultured tumor cell lines (VCaP, LNCaP, or a CTC line derived from a breast cancer patient) were spiked into healthy donor blood samples at 2000-3000 cells per milliliter of blood. After that, the blood sample was divided into appropriate volumes and treated with the platelet inhibitors tirofiban (0.5 or 1 ⁇ g/mL) or eptifibatide (20 ⁇ g/mL), before they were immediately stored or proceed to microfluidic processing. Samples were stored under room temperature or 4 °C, protected from light and without rocking. The platelet inhibitor cocktail included EDTA (2-5 mM) which was added to the blood samples 15 minutes prior to microfluidic processing. 5-6 mL of blood samples were processed per experimental condition.
  • the blood first passes through a filtration array which removes large aggregates and then reaches the hydrodynamic sorting stage which removes plasma, platelets, and red blood cells based on size.
  • the enriched nucleated cells were then aligned into a single line such that the Dynabead-targeted leukocytes were effectively depleted by magnetophoresis.
  • the enriched tumor cells underwent a second hydrodynamic sorting array which removes residual platelets and red blood cells. The remaining highly enriched tumor cells were then collected in the product outlet in PBS buffer containing 1% Pluronic. Enumeration of spiked cells and carryover of leukocytes was performed according to published protocols ( Karabacak et al. 2014. Nat. Protoc. 9(3):694-710 ).
  • a breast CTC line labeled with luciferase was used for the quantification of cell growth ( Yu et al. 2014. Science. 345(6193):216-20 ).
  • the blood sample was spiked with 3000 CTCs/mL of blood, and 6 mL of blood was processed by the iChip.
  • the enriched CTC product was then spun down, resuspended in 2 mL of CTC culture media (Yu et al. 2014), and cultured in low-adhesion 24-well plates at 500 uL per well (4 wells total). On the day of assay, 400 uL of cells from the well and the number of viable cells was determined using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's protocol.
  • Luminescence signal was measured using a SpectraMax M5 Microplate Reader (Molecular Devices).
  • a positive control cells from the same CTC line was cultured directly and assayed with the same method, without any spiking processes in blood or iChip sorting. These cells were adjusted to 4500 cells in 500 uL media and cultured in the same plates.
  • EL Buffer QIAGEN
  • Platelets were tested at various temperatures in order to understand the relationship between cold storage and platelet activation. These experiments were also performed in order to identify the best temperature for storage whereby platelet activation could be minimized with maximal preservation of whole blood components.
  • CD62/p-selectin and GPIIa/IIIa play a critical role in platelet aggregates which clog microchannels of the CTC-iChip.
  • CD62/p-selectin is associated with degranulation - a process that leads to the recruitment and activation of surrounding platelets; suggesting inhibition would impede signaling to nearby platelets and minimize signal amplification.
  • activated GPIIb/IIIa and CD62/p-selectin mediate platelet-platelet, platelet-leukocyte, and platelet-CTC interactions, and interact with the microfluidic device itself, resulting in improper cell sorting, clogging, and slow flow rates.
  • platelet inhibitors including Tirofiban (Sigma), eptifibatide (Tocris), Clopidogrel (Sigma), KF38789 (Tocris).
  • Echinocytes are commonly observed in ex vivo blood samples and are an indicator of cellular stress. Echinocytes present major complications for microfluidic applications that rely on the specific size, shape and flexibility of the cells to enable efficient, rapid sorting. As erythrocytes account for 99% of blood volume, these structural changes significantly interfere with microfluidic processes in CTC isolation, as these spherical echinocytes are deflected like nucleated cells in hydrodynamic cell sorting platforms.
  • cold storage can preserve the morphology of up to 80% of RBCs. Also, the addition of the platelet inhibitor cocktail (tirofiban plus EDTA) does not have any deleterious effect on echinocyte formation
  • RNA integrity was enhanced in cold (Example 3.6, Fig. 15 ). Since RNA is one of the most sensitive biological materials, we view this as a robust marker of preservation. The results showed that RNA integrity at 48hr storage was similar for room temperature and cold stored blood, and that after 72hr storage, cold stored RNA remained intact, while room temperature stored blood was severely degraded.
  • Platelet inhibitors enabled CTC-iChip processing of both fresh blood and blood that was stored for 3 days under hypothermic temperature 4 °C.
  • the platelet inhibitor (PI) used was either 0.5 ⁇ g/mL tirofiban, or 1 ⁇ g/mL tirofiban + eptifibatide 20 ⁇ g/mL.
  • EDTA (2-5 mM) was added to blood samples 15 minutes prior to iChip processing. The results showed the following: Clogging of the iChip in the absence of platelet inhibitors or as a result of storage for 72 hours in room temperature (RT) led to severe clogging in the first stage of the filtration array.
  • the image in Fig. 17A shows fluorescence staining of DNA by Vybrant DyeCycle Green (Life Technologies) which illustrates cells trapping in the clog. Scale bar represents 100 ⁇ m.
  • FIG. 17B The plot illustrates the blood volume that was processed as a percentage of the target volume (5-6 mL). In the presence of platelet inhibitors and EDTA, ⁇ 90% of blood volume could be processed; the residual volume losses was attributed to sample transfer and dead volumes in tubing and is standard in microfluidic chips.
  • Fig. 17E The log-transformed fold depletion of leukocytes by the iChip is shown in Fig. 17E .
  • a depletion of 4 log translates to 10000-fold depletion, which means that a typical blood sample containing 5 ⁇ 10 6 leukocytes /mL of blood would leave only 500 leukocytes /mL of blood processed in the enriched CTC product.
  • the use of platelet inhibitors and EDTA greatly improved depletion, presumably by preventing platelet-leukocyte interaction ( Fig. 12 ) and thereby allowing access of leukocyte-depletion antibodies. Blood stored for 72 hours in room temperature also suffered from low depletion, possibly as a result of leukocyte degradation ( Fig. 15 ).
  • Fig. 17F shows carryover of red blood cells (RBC) / ⁇ L of blood into the CTC product.
  • RBC red blood cells
  • Room temperature-stored blood samples showed higher RBC carryover presumably due RBC degradation, which changes shape and form echinocytes ( Fig. 13 ) and were unable to be removed effectively in the hydrodynamic sorting stage of the iChip.
  • Luminescence signal was normalized to the signal at day 0 which was the day when culture was initiated immediately after iChip processing. A positive control was included without any iChip processing.
  • CTCs sorted from a fresh blood sample (0 hr) in the presence of platelet inhibitors exhibited growth rates almost identical to the positive control, suggesting that the microfluidic processing and treatment with platelet inhibitors and EDTA were non-toxic to CTCs.
  • CTCs could be sorted and re-cultured in vitro, although there was a slight delay in re-initiating proliferation.
  • CTCs obtained from a blood sample stored for 72 hours in room temperature showed a decreased ability to proliferate after sorting, as compared to CTCs stored at 4°C.

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